Thiocarlide (THC), a thiourea, inhibits the synthesis of mycolic acids and fatty acids in Mycobacterium tuberculosis. Our evidence indicates that the main effect of THC on fatty acid synthesis is in inhibiting the synthesis of oleic acid, directly attributable to the inhibitory effect of THC on the activity of the membrane-associated stearoyl-CoA (delta9) desaturase DesA3 (Rv3229c). Sterculic acid, a known inhibitor of delta9 desaturases, emulates the effect of THC on oleic acid synthesis but does not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. Therefore, THC has at least one other enzymatic target in the mycolic acid biosynthetic pathway. Since our preliminary data also indicate that THC has no inhibitory effect on the activity of the FAS-I and FAS-II systems from Mycobacterium aurum, the second target of the drug could be an enzyme introducing functional groups into the meromycolate chain of mycolic acids such as a membrane-associated desaturase. Bioinformatics has revealed four putative membrane desaturases with the characteristic His-rich motif, all potential targets of THC/thioureas, as well as two soluble-type desaturases. Moreover, the genome of M. tuberculosis potentially encodes eleven proteins structurally related to mammalian-type epoxide hydrolases which are known in eukaryotes to be sensitive to urea inhibitors. All of these enzymes will be over-expressed and minimal inhibitory concentration to the THC compared. In a broader genetic approach, mutations in the genome of mycobacteria responsible for conferring high level resistance to THC will be characterized. The essentiality, function and 3D structure of the M. tuberculosis desaturases and newly identified target(s) of THC will be established and cell free assays/screens developed to a low screening of several libraries of inhibitors including our existing library of THC derivatives, facilitate a medicinal chemistry approach in the form of chemical synthesis and extended testing of products. THC was once acceptable for treatment of tuberculosis but lost favor due to untoward absorption kinetics and low bioavailability. This approach may overcome these aspects.